Abstract

We investigate the limits of one-photon fluorescence as a contrast mechanism in nanoscale-resolution tip-enhanced optical microscopy. Specifically, we examine the magnitude of tip-induced signal enhancement needed to resolve individual fluorophores within densely-packed ensembles. Modulation of fluorescence signals induced by an oscillating tip followed by demodulation with a lock-in amplifier increases image contrast by nearly two orders of magnitude. A theoretical model of this simple modulation/demodulation scheme predicts an optimal value for the tip-oscillation amplitude that agrees with experimental measurements. Further, as an important step toward the eventual application of tip-enhanced fluorescence microscopy to the nanoscale structural analysis of biomolecular systems, we show that requisite signal enhancement factors are within the capabilities of commercially available silicon tips.

(Color Online) Expected phase dependency of lock-in signal. Each detected photon is considered as a unit vector with a direction corresponding to the instantaneous oscillation phase of the tip. A lock-in amplifier performs the vector addition of all such unit vectors. The near-field photon phases are Gaussian distributed around θp, which corresponds to tip-sample contact. Far-field background photons are detected randomly at all phases so the corresponding vector addition is simply a random walk.